The broad, long-term objectives of the research proposed is to fully characterize a novel metabolic pathway expressed in both macrophages and endothelial cells. The studies proposed here will be carried out exclusively with macrophages. This pathway, oxidation of L-arginine to N=O, NO2- and NO3-, plays an integral role in two important biological processes, namely macrophage cell- killing activity and vasodilation. This characterization will include the isolation and purification of the enzyme or enzymes involved in the pathway, kinetics of the various reactions and chemical characterization of the intermediaries in the reaction. The enzyme or enzymes involved the oxidation of L-arginine to NO2- and NO3- will be purified to homogeneity. The assay that is used presently measures NO2- and NO3- which are the end products of the reaction. Therefore, it will be consumption, and L-citrulline formation. The newly isolated enzymes will be characterized. This will include basic kinetic experiments to measure rates and substrate(s) affinity for any of the individual enzymes isolated. It may be that the reaction is carried out by a multienzyme complex, and if that is the case this protein will be characterized in similar manner. Substrate specificity intermediates in the reaction of L-arginine and NO2- and NO3- will be carried out. The intermediaries known at this time are N-hydroxy-L-arginine and N=O. Studies will involve stability of any intermediates isolated and the chemistry by which N=O is generated from L-arginine. Methods of protein purification, synthetic organic chemistry, an analytical biochemistry will be brought to bear on the overall problem. A complete understanding of the regulation and enzymology of this pathway should lead to national control over processes as diverse as immune system status and vasodilation.